1. Field of the Invention
The present invention relates to fed-batch fermentation, and more particularly to computer automated feed-back control of the nutrient level of a broth in fed-batch fermentation.
2. Description of the Prior Art
During fermentation processes, the bacteria or yeasts growing in a fermentation broth consume nutrient at a variable rate related to, among other things, the microorganism density and rate of growth. In the case of fed-batch fermentation of bacteria, for example, the rate of consumption of nutrient, typically, glucose, can increase exponentially with time until affected by the limitations of the environment or alteration of the conditions, such as varying the rate of agitation and aeration. Another process interference results from the introduction of chemical agents for inducing the bacteria to produce recombinant DNA products. Accordingly, the yield or productivity of a fermentation process is increased when nutrient is added during the fermentation to compensate for that depleted through consumption by the bacteria.
It is desirable to maintain a constant nutrient concentration throughout the fermentation process despite the variable rate at which the nutrient is depleted. When nutrient concentration, usually glucose, is very high, undesirable waste by-products, usually acetic acid, lactic acid or ethanol are produced. The economic implications of inefficient nutrient utilization are very important because of the high cost of glucose. When the nutrient concentration is too low, or absent, the growth of the microorganisms is restricted usually resulting in reduced productivity of the process. Thus, significant efforts have been expended in attempting to develop methods for maintaining the nutrient concentration relatively constant during the fermentation process. Nevertheless, completely satisfactory techniques have not been found to maintain the concentration within a sufficiently desirable narrow range, especially in the situations in which the standard exponential consumption rate is disrupted.
Generally, manual techniques have been employed for controlling the nutrient concentration by measuring the nutrient level of the medium and replenishing the nutrient to compensate for depletion. Recent reports have described the development of at least partially automated techniques. For example, in G. Luli et al., "An Automatic, On-Line Glucose Analyzer for Feed-Back Control of Fed-Batch Growth of Escherichia coli", Biotechnology Techniques, Vol. 1, No. 4, pp. 225-230 (1987), a process control technique for maintenance of glucose concentration is described in which the glucose level is monitored periodically and matched against archived profiles of glucose consumption rate versus time as determined by earlier experimentation. The amount of glucose to be introduced during the next interval is then determined according to the archived curve. This process also required the separation of cells from the broth by membrane filtration prior to analysis of the cell-free medium for nutrient concentration. Glucose concentrations were maintained between 1.0 and 2.0 grams per liter with this method.
In a later paper, G. Lull et al., "Comparison of Growth, Acetate Production and Acetate Inhibition of Escherichia coli Strains in Batch and Fed-Batch Fermentations", Applied and Environmental Microbiology, April 1990, pp. 1004-1011, a similar technique with a higher sampling rate is discussed. The article reports that archived data for glucose consumption rates were required for computer-controlled glucose addition. The glucose concentration is reported to have been maintained at about 1.0+/-0.2 g/l.
G. Kleman et al., "A Predictive and Feedback Control Algorithm Maintains a Constant Glucose Concentration in Fed-Batch Fermentations", Applied and Environmental Microbiology, April 1991, pp. 910-917, describes a method which requires linear regression analysis of nutrient concentrations to feed-forward control the addition of nutrient to match consumption rate (glucose demand, GD). The method assumes that the theoretical glucose demand is based on a constant yield of biomass from glucose. The method requires cell-free broth for analysis of nutrient concentration requiring frequent broth sampling at two minute intervals and has a response time between sample analysis and nutrient pump response.
However, such techniques suffer from several drawbacks. The technique of Luli et al. requires that numerous trials of the particular strain of microorganism under various conditions and desired nutrients and nutrient concentrations be conducted to prepare an archive of nutrient consumption rate curves for comparison purposes. In addition, because the nutrient feed rate is dependent on the archived curve, a curve for the same strain being cultivated under the same conditions must be located in order to predict the rate of consumption of the nutrient during the next time interval. Further, if the fermentation conditions change, for example, if the agitation rate is varied or if a chemical agent is introduced to induce the microorganism to produce recombinant DNA products, archived curves cannot be relied on. The requirement for cell-free broth for nutrient analysis adds another level of complexity to the method. Although the second Luli et al., article makes reference to control of glucose concentration at 1.0 gram per liter +/-0.2 grams per liter, it appears that such control is maintained only for undisturbed fermentation conditions with standardized strains of Escherichia coli. Again the major limitations of this method is that this system does not adapt to variances from the conditions under which the archived consumption rate curves were derived, and cell-free broth is required for nutrient analysis. Kleman et al., requires a linear regression analysis in the algorithm and is therefore a major limitation to the method. When glucose consumption rates are very high the method significantly underpredicts glucose demand. Further, linear regression analysis for determining glucose demand during metabolic shifts creates errors in response to matching glucose demands and feed rates.